Separating materials for linking TLC and FTIR

ABSTRACT

The invention concerns separating materials for thin-layer chromatography, the materials having a sorbent layer formed from sorbent particles and disposed on a carrier. A reflection intensifier, preferably magnesium tungstate and/or Bi 2  O 3 , is added to the sorbent layer. This addition comprises at least 10 wt % relative to the amount of sorbent. In preferred embodiments, the particle size of the reflection intensifier is 3 to 5 μm and the amount of the reflection intensifier is 0.6 to 15 times the amount of sorbent. These separating materials are particularly suitable for the in-situ measurement of FTIR spectra. The invention also concerns the use of these separating materials for the in-situ measurement of FTIR spectra.

The invention relates to separating materials for thin-layerchromatography (TLC) which are suitable, in particular, for in situinfrared spectroscopy (IR) and Fourier transform infrared spectroscopy(FTIR).

For some time now research has been carried out on linking TLC toinfrared spectroscopy, the objective being to combine TLC, a powerfuland economical separation method, with IR, a spectroscopic methodacknowledged as being informative (Percival C. J., Griffiths P. R.(1975), Anal. Chem., 45 (1), pp. 154-156). Despite the major effort infabricating special layers (e.g. silver chloride as the base, with anaddition of the perfluorinated hydrocarbon fluorolube to reducescattering) the detection limit and sensitivity remained inadequate.Moreover, if silica gel was used as the stationary phase, the spectralrange important for analytical purposes and known as the "fingerprintrange" could not be used, owing to the intrinsic absorption of thesilica gel.

Owing to its silanol and siloxane groups, silica gel is one of thestrongly absorbing materials. Reflectance spectroscopy with suchstrongly absorbing materials involves an anomalous profile of thedispersion curve, most of the incident radiation being reflecteddirectionally. This leads to so-called "residual rays" which no longercontain any spectral information. Further problems arise from the factthat the effective penetration depth in the vicinity of absorption bandsof the matrix is much less than in spectral ranges without matrixabsorption. Instead of silica gel, Danielson et al. (1992) Analyt. Chem.64, 2183-2186, therefore employed zirconium oxide as the sorbent. Whileit was possible to measure FTIR spectra of substances adsorbed on thissorbent, many substances which can be separated without any problems onsilica gel were impossible to separate on zirconium oxide, since theyeither remained in the start zone or migrated with the solvent front.This meant that the analytically important range of R_(f) values between0.2 and 0.8 could not be attained.

In the wave number range between 1350 and 1000 the strong intrinsicabsorption of the silica gel in this range produces a strong interferingband which is superimposed on the DRIFT spectra (diffuse reflectanceinfrared Fourier transform; reflectance spectra in the medium-infraredrange) of adsorbed substances. Consequently, significant portions of the"fingerprint range" can no longer be covered. DRIFT measurements onsilica gel layers were first carried out by Fuller and Griffiths (FullerM. P., Griffiths P. R. (1978), Anal. Chem. 50 (13), pp. 1906-1910; andFuller M. P., Griffiths P. R. (1980) Appl. Spectrosc., 1980 (34), pp.533-539). In so doing, spectra of methylene blue could be measured with1.2 μg of substance.

Given the versatility of the possible applications, in particular ofsilica gel 60 (pore size 60 Angstroms) as a sorbent in TLC and HPTLC andthe quality standard achieved, in particular, for this stationary phasethere is a need for the development of suitable precoated layers on thebasis of silica gel for linking with IR spectroscopy. It is an object ofthe invention to provide separating materials for thin-layerchromatography which permit an in situ study by means of FTIRspectroscopy even with small amounts of sample material. This object isachieved by the provision of separating materials for thin-layerchromatography, whose sorbent layer comprises an addition of areflection enhancer.

The invention relates to separating materials for thin-layerchromatography which comprise a sorbent layer, formed from sorbentparticles, on a support, the sorbent layer comprising an addition of atleast 10 percent by weight, based on the amount of sorbent, of areflection enhancer, preferably magnesium tungstate and/or Bi₂ 0₃. Inpreferred embodiments the particle size of the reflection enhancer is3-5 μm and the amount of the reflection enhancer is from 0.6 to 1.5times the amount of sorbent. SiO₂ -containing sorbent particles arepreferred.

The invention further relates to the use of this separating material forseparation methods followed by in situ measurement of an FTIR spectrum.

Reflection enhancers suitable according to the invention have goodreflectance in the IR. Such materials are known in principle. Theyinclude metals, insofar as they are resistant to the eluents customaryin thin-layer chromatography, and oxygen-containing metal compounds. Inparticular, according to the invention preference is given to magnesiumtungstate and Bi₂ O₃ as reflection enhancers. According to the inventionit is also possible for a plurality of substances in mixed form to beadded as reflection enhancers. These also include compounds, e.g.magnesium tungstate, which were added as a fluorescent indicator insmall amounts (about 2 percent by weight) to the known sorbent layers.According to the invention, however, the reflection enhancer content ismore than 10 percent by weight, based on the amount of sorbent.

The addition, according to the invention, of a reflection enhancer tothe sorbent layer of silica gel preserves the good chromatographicproperties of the sorbent; nevertheless, the signal-to-noise ratio (e.g.in recording a Gram-Schmidt chromatogram, which involves summation overthe entire IR spectral range) and the quality of the DRIFT spectra areimproved. This improvement in the IR spectroscopic characteristics isalso manifested, for example, by the disappearance of the interferingband at wave number 1337.

The grain size and grain size distribution of the reflection enhancersused according to the invention is in the same order of magnitude as thesorbent particles used in TLC or HPTLC. Preferably the grain size of thereflection enhancers is in the range of about 2-20 μm, in particular ofabout 3-5 μm.

The quantitative proportion of the reflection enhancer in the sorbentlayer is more than 10 percent by weight, preferably from 0.1 to 9 times,particularly preferably from 0.6 to 1.5 times the amount of sorbent.

Sorbents to be considered include the sorbents customarily used inthin-layer chromatography, in particular silica gel and derivatizedsilica gels. A large number of derivatized sorbents are known to thoseskilled in the art and are mentioned in standard publications. Theseinclude, in particular, the hydrophobic reversed phase materials knownas C₂, C₈ and C₁₈, and hydrophilic materials modified with amino, cyanoor diol groups.

As a further addition the sorbent layer may also comprise fluorescentindicators customarily used in thin-layer chromatography. Sorbents andfluorescent indicators are known to those skilled in the art.

Separating materials for thin-layer chromatography normally additionallycomprise binders. Binders suitable in principle and their proportions inthe sorbent layer are known to those skilled in the art. Inasmuch asbinders are used for the novel separating materials, particularpreference is given to poly(meth)acrylic acid and poly(meth)acrylates.Particularly preferred binders have mean molecular weights of 3-4million and are commercially available.

The sorbent particles of the novel separating materials may be of bothirregular and regular shape and in particular may be spherical.

The support used for the separating layer can take the form of the glassplates or films customarily used in TLC or HPTLC, but alsosurface-treated supports, e.g. roughened supports, mirrored supportsand/or supports metallized by vapor deposition.

The layer thickness of the sorbent layer of the novel separatingmaterials corresponds to the layer thicknesses customary in TLC andHPTLC. Layer thicknesses of between 30 and 250 μm, in particular between50 and 140 μm, are preferred according to the invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows Gram-Schmidt chromatograms of phenazone, caffeine andparacetamol on a sorbent layer according to the invention, comprising50% by weight of Bi₂ O₃, compared with a layer without reflectionenhancer.

FIG. 2 shows the DRIFT spectrum, recorded in situ, of caffeine on asorbent layer according to the invention, comprising 50% by weight ofBi₂ O₃, compared with a layer without reflection enhancer.

Details on both figures can be found in Example 2.

FTIR spectrometers are commercially available. The in situ measurementof samples separated by thin-layer chromatography typically makes use ofan external TLC/HPTLC DRIFT unit. Such units are likewise commerciallyavailable; for example, given a fixed measuring beam the thin-layersupport is moved on a stage with X and Y movements by means of stepmotors. The measuring setup is typically purged with dried, CO₂ -freeair. Control of the movement and data acquisition, and processing of themeasurement data are carried out by a computer, with the option, ifrequired, of background spectra being measured on an empty point of thethin-layer plate and being subtracted in the course of data processing.Suitable computers are commercially available, as are control andevaluation programs.

EXAMPLE

The following examples are intended to illustrate the subject matter ofthe invention without limiting the inventive idea.

Example 1 Preparation of a HPTLC plate on the basis of silica gel 60 forlinking to FTIR

a) Preparation of the suspension

320 mL of water are admixed with 2.0 g of a high molecular weightacrylic acid (mean molecular weight 4×10⁶), which are stirred in for 15min at about 1200 rpm. The solution is admixed with 50 g of a silica gel60 (mean particle size about 5 μm; grain size distribution 4-8 μm)customarily used in HPTLC, 50 g of a Bi₂ O₃ powder (mean particle sizeabout 4 μm) and 2.0 g of Mg tungstate (mean particle size about 3 μm),followed by stirring for a further 15 min at about 1200 rpm. Using 0.1Nsodium hydroxide solution, the suspension is then set to a pH of 6.3,followed by stirring for a further 1 h at about 1200 rpm.

b) Coating of the glass plates

Glass plates of format 20×20 cm (glass thickness 1.25 mm) are coated asfollows with the prepared suspension: A Desaga coater is charged withthe suspension and passed across the glass plates, the knife being setto about 0.25 mm.

c) Drying

The coated glass plates are dried in a drying cupboard at about 110° C.for 30 min. The resulting layer thickness is about 100 μm.

Example 2 Separation of substances and ensuing linking to FTIR

a) Separation of substances

5 μL of a solution, 0.02% strength in each case, of caffeine,paracetamol and phenazone in dichloromethane are sprayed onto the plate(using Linomat IV from CAMAG) in the form of bands (band length 3 mm).After drying the plate is developed in a normal chamber, without chambersaturation, with the mobile phase ethyl acetate/methanol 20/1 (v/v) to amigration distance of 4 cm and is then dried.

b) Recording of spectra

The measurements are carried out using an IFS48 FTIR spectrometer andthe external DRIFT unit from Bruker. Evaluation is carried out via thespectrometer software OPUS Vers. 4.17 A.

The results are summarized in FIGS. 1 and 2:

FIG. 1 shows Gram-Schmidt chromatograms of phenazone (1), caffeine (2)and paracetamol (3) on a sorbent layer according to the invention,comprising 50% by weight of Bi₂ O₃ (continuous line), compared with alayer without reflection enhancer (broken line).

FIG. 2 shows the DRIFT spectrum, recorded in situ, of caffeine on asorbent layer according to the invention, comprising 50% by weight ofBi₂ O₃ (continuous line), compared with a layer without reflectionenhancer (broken line). (1) marks the position of the interfering bandat wave number 1337 cm⁻¹, which occurs only in the comparative spectrum.

We claim:
 1. A separating material for thin-layer chromatography which comprises, on a support, a sorbent layer comprising sorbent particles and at least 10 percent by weight, based on the weight of sorbent particles, of magnesium tungstate and/or Bi₂ O₃ as a reflection enhancer.
 2. The separating material according to claim 1, wherein the reflection enhancer is in the form of particles having a particle size of 2-20 μm.
 3. The separating material according to claim 1, wherein the amount of the reflection enhancer is from 0.1 to 9 times the amount of the sorbent particles.
 4. The separating material according to claim 1, wherein the sorbent particles comprise SiO₂.
 5. The separating material according to claim 1, wherein the sorbent particles are hydrophobic reversed phase materials or hydrophilic materials modified with amino, cyano or diol groups.
 6. The separating material according to claim 1, wherein the separating material further comprises a high molecular weight acrylic and/or methacrylic acid polymer as a binder.
 7. The separating material according to claim 1, wherein the thickness of the sorbent layer is between 30 and 250 μm.
 8. The separating material of claim 1, wherein the reflection enhancer is in the form of particles having a particle size of 3-5 μm.
 9. The separating material of claim 1, wherein the amount of the reflection enhancer is from 0.6 to 1.5 times the amount of the sorbent particles.
 10. A thin-layer chromatography method which comprises separating components of a composition on a separating material according to claim 1 and measuring, in situ, the FTIR spectrum thereof. 